Protein Production in E. coli

ReliaTech offers a modular system of customer services for gene expression and protein production in E. coli. ReliaTech's “E. coli Expression Service” is divided into a series of steps which can be performed either individually or in combination - so you can decide which of the steps you actually need.

These services are designed to adequately cover all specific demands along the line from gene to protein, e.g. gene cloning into an E. coli expression vector, screening for the best vector and/or E. coli strain, production of biomass, as well as protein purification.

Along with the regular project updates, you will receive progress reports at the end of each service step. We will guide you from step to step dependent on your authorization for us to proceed.

The success of this step is always dependent on the gene which shall be expressed. Taking into account your specific requirements, ReliaTech can perform all essential experiments, including gene modification, prior to insertion into an expression vector.

Alternatively, assistance is provided to the customer allowing him to choose a suitable expression vector and the right conditions to ensure the optimal gene expression.

Because E. coli strains are not able to cleave the signal peptide of secreted proteins the cDNA has to be modified. Therefore the cDNAs are normally generated by PCR deleting the sequence for the signal peptide and introducing the appropriate restriction sites for cloning (in many E. coli expression vectors: NdeI and BamHI).

Option A1 - Standard Cloning

The construct has to contain the appropriate restriction sites for subcloning (e.g. NdeI/BamHI, for further information, please inquire).

Our service comprises:

Subcloning of the cDNA into the E. coli expression vector, e.g. pET-9a, pET-15b, pET-21b or pCytexP3 or into an expression vector chosen by you.

Plasmid-preparation of 10 recombinant clones.

Analysis by agarose-gel electrophoresis.

Mid-scale plasmid preparation of a positive clone.

Please, note: Appropriate restricitons sites are mandatory for using this service option. To make sure using the proper sites either inquire for further specification or use our PCR cloning service (see below).

Results provided to you

An E. coli expression vector containing the cDNA of interest.

A detailed report sheet.

Expected time range: 2-4 weeks

Option A2 - PCR Cloning

Material we need from you:

10-30 μg purified and characterized plasmid cDNA bearing the gene to be expressed.

Subcloning of the PCR-fragment in the E. coli expression vector e.g. pET-9a, pET-15b or pCytexP3 or in an expression vector chosen by the customer.

Plasmid-preparation of 10 recombinant clones.

Analysis by agarose gel electrophoresis.

Mid-scale plasmid preparation of a positive clone.

Verification by sequencing.

Note: All cDNA’s generated by PCR have to be completely sequenced due to possible mutations!

Results provided to you:

an E. coli expression vector containing the cDNA of interest.

A detailed report sheet.

Expected time range: 4 - 6 weeks

PCR cloning - Recommendations

For an optimal performance of a new protein it is recommended to test different E. coli strains and E. coli expression vectors either induced by a temperature shift or by IPTG.

With respect to the purification procedure we would recommend introducing a “tag” at the N- or C-terminal end of the recombinant protein. There are E. coli expressions vectors available containing different “tags”. If the recombinant protein is used mainly in in vitro experiments (e.g. cell culture studies) we recommend for example a “His-tag” (6x Histidin) or a “Strep-tag II” (8 amino acids) [www.iba-go.de]. The “tag” is normally too small to interfere with the activity of the protein. For “Strep-tag II” the possibility exists to create an authentic protein by cleavage of the tag. If the recombinant protein is needed for animal studies (e.g. mouse, rat) we recommend the use of an “Fc-tag” (about 26 kDa) to increase the stability and half-life of the protein in the circulation. As an “Fc-tag” ReliaTech can offer human IgG1 and the mouse IgG2b fragments.

This step enables you to produce your own recombinant E. coli strain for your individual production purposes.

Material we need from you:

10-30 μg purified and characterized E. coli expression vector DNA bearing the gene to be expressed. The vector is either provided by you or prepared by us (for further information concerning our cloning services, please click here).

Note: For the optimal performance of a new protein it is recommended to test the bacterial host by using several different E.coli strains for the analytical production, e.g. BL21 (DE3), BL21 Star (DE3), Rosetta (DE3) and others (please, inquire).

Our service comprises:

Generation of a recombinant E.coli strain using standard protocols.

Isolation and cultivation of 6 single clones over night.

Analytic production (1ml scale)

SDS-PAGE and subsequent Coomassie stain of total lysate from induced/non-induced samples.

The induction of our standard constructs for protein expression is performed by temperature shift or IPTG.